G L A S E R A N D LI
Reaction of Bovine Growth Hormone with Hydrogen Peroxidet Charles B. Glaser and Choh Hao Li*
ABSTRACT : Methionine residues in bovine pituitary growth hormone were selectively modified with hydrogen peroxide in Tris buffer at pH 8.5 at 25". The results indicate that three of the four methionine residues were oxidized. The nonoxidized residue was found to be located at position 179 along the polypeptide chain. The oxidized protein was compared to the native hormone as to the relative rates of trypsin digestion,
A
previous study from this laboratory reported on the specific modification of tyrosyl residues in bovine and sheep pituitary growth hormones with tetranitromethane (Glaser et al., 1973). It was shown that of the six tyrosyl residues present in each molecule, two were completely modified, two were partially modified, and two were unreacted at identical sites along the linear chain. Various physical, chemical, and biological data showed the close structural similarity of the two modified hormones, both in relation to their parent compounds and to each other. A study of the accessibility of methionine residues to hydrogen peroxide under various solvent conditions was undertaken in the present work. Several related investigations on protein hormones have been previously reported. In bovine parathyroid hormone (Tashjian et al., 1964), pig adrenocorticotropin (Dedman er al., 1957, 1961 ; Dixon and Stack-Dunne, 1955), and human calcitonin (Neher et al., 1968), oxidation of methionine residues to the corresponding sulfoxide derivatives was accompanied by significant losses in biological function. In the first two examples cited, restoration of biological activity was achieved via reduction with thiol reagents. In addition, the report on bovine parathyroid hormone presented immunological evidence that the loss in biological activity was not accompanied by major conformational alterations. The subject of this report is the selective oxidative of methionine residues in BGH1 and the effects on functional and structural parameters. Materials and Methods The pituitary growth hormones from beef (Li, 1954) and sheep (Papkoff and Li, 1958) pituitaries were isolated according to methods previously described. Each was further purified on Sephadex G-100 (Pharmacia) in 0.05 M Tris buffer (pH 8.5). Glycine and 2-amino-2-(hydroxymethyl)-l,3-propanediol were obtained from Eastman Organic Chemicals, Rochester, N. Y . , and used without further purification. Catalase was purchased from Sigma Chemicals, St. Louis, Mo. Iodoacetic-l-14C acid was obtained from International Chemical and Nuclear Co., Irvine, Calif. Urea was purchased
t From the Hormone Research Laboratory, University of California, San Francisco, California 94143. Received August 27, 1973. This work was supported in part by the American Cancer Society, the Allen Foundation, and the Geffen Foundation. 1 Abbreviations used are: BGH, bovine pituitary growth hormone; SGH, ovine pituitary growth hormone; CD, circular dichroism.
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behavior in exclusion chromatography, circular dichroism, ultraviolet difference and fluorescence spectroscopy, and biological activities. The data show that the oxidation is specific, and that both structural and functional parameters are preserved. It was also shown that the pattern of methionine oxidation in sheep pituitary growth hormone on peroxide treatment is identical with that of the bovine hormone.
from Mallinckrodt Chemical Co., St. Louis, Mo., and was recrystallized from 95% ethanol before use. Dioxane was obtained from Matheson Coleman and Bell, East Rutherford, N. J. Toluene-14Cwas received from Packard Instrument Co., Downers Grove, Ill. All other chemicals were reagent grade. Deionized water was used throughout. Kinetics of the Oxidation Reaction. The protein was dissolved (4 mg/ml) in the appropriate solvent containing 0.0005 M EDTA, and hydrogen peroxide (30%) was added to a final concentration of 0.3 M. The solution was stirred at 25 '. At various times, 0.2-ml aliquots were removed and added to a solution containing 333 units of catalase in 1 ml of phosphate buffer at pH 7.0; one unit of the enzyme will hydrolyze 1 pmol of H*02/minat pH 7.0 and 25". After -10 min the solution was dialyzed against 0.001 N HCIOl and lyophilized. Specific alkylation of the nonoxidized methionine residues was achieved by the addition of 2.75 ml of 8 M urea (pH 3.5) containing 15.1 mg o f i ~ d o a c e t i c - l - ~ ~ C a(sp c i dact. = 4.45 x 10-2 Ci/mol) according to the procedure of Wallis (1972). After 48 hr at 37", the reaction was terminated by extensive dialysis against 0.1 N glycine-HC1 buffer of pH 3.6 until the radioactivity in the outside solution was reduced to that of buffer alone. After measuring the protein concentration of each solution, its radioactivity was determined on a Packard Model 3320 Tri-Carb liquid scintillation spectrometer as follows: 0.5 ml of the protein solution in the glycine buffer was added to 10 ml of scintillant mix (1 1. of dioxane, 143 g of naphthalene, 4.5 g of 2,5-diphenyloxazole, and 0.58 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene).The counting efficiency in this system was determined using toluene-14Cas a standard and found to be 78 %. Preparation of Peroxide-Oxidized BGH. The protein (120 mg, 4.74 pmol) was dissolved in 25 ml of Tris buffer (0.05 M, pH 8 . 5 ) containing 0.0005 M EDTA at 25". Hydrogen peroxide was added to a final concentration of 0.3 M. The reaction mixture was stirred for 5 hr and then submitted to gel filtration on a Sephadex G-25 column. After dialysis and lyophilization of the unretarded material, the dimeric product was separated from higher molecular weight aggregates by exclusion chromatography on Sephadex G-100 (3 X 68 cm) in the same buffer. After dialysis the product was obtained on lyophilization. Amino acid analyses, rate of tryptic digestion, circular dichroism, and ultraviolet fluorescence spectra were performed by procedures previously described (Glaser et a/., 1973). The protein concentration of the unmodified hormone c was obtained using a value E: $",2~7 nm = 7.30 as determined
OXIDATION OF BOVINE GROWTH HORMONE
previously (Bewley and Li, 1972). The same value was employed for peroxide-oxidized derivatives of BGH and SGH. Autoradiographic Procedure for Identification of Nonoxidized Methionine. Peroxide-oxidized BGH . (3.0 mg) was allowed to react with 3.0 mg of iod~acetic-Z-~~Cacid (sp act. = 3.4 x 10-1 Ci/mol) as described above. The product was dissolved in 0.1 M NH40Ac of pH 8.5 and digested with trypsin (enzyme/substrate ratio, 1/25) for 24 hr ;the lyophilized material was submitted to two-dimensional chromatographyelectrophoresis. Chromatogtaphy was performed on Whatman No. 3MM paper with a solvent system consisting of nbutyl alcohol-acetic acid-water (4 :1 :5, v/v), and high voltage electrophoresis in formic acid-acetic acid-water buffer of pH 2.1 for 1 hr at 2000 V (Gilson high voltage electrophoretor, Model D). The chromatogram was spotted in the corners with a solution of iodoacetic-l-14C acid, put into direct contact with Du Pont Cronex X-ray film for 60 hr and developed with a Kodak RP-x-omatic processor. Only one radioactive spot was visible on the film, Treatment of the peptide map with ninhydrin gave a positive response corresponding exactly to the radioactive spot. After elution with 0.1 N N H 4 0 H the solution was lyophilized and hydrolyzed for amino acid analysis. Bioassay. The growth promoting potency of the modified protein was estimated by the rat tibia test (Greenspan et a[., 1949).
Amino Acid Analyses of Native and HzOz-Oxidized Bovine Pituitary Growth Hormone.
TABLE I:
Native
HpOl-Treated BGH
Amino Acid
BGH
pH 8.5*
pH 2.85'
Tryptophana Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Methionine sulfone
1 10.4 3.1 12.2 16.6 13.5 11.4 22.9 5.8 11.2 12.8 3.6 6.4 3.9 7.6 26.2 5.9 12.4 0
1 9.8 3.1 10.8 16.6 12.0 11.6 23.6 6.5 11.2 13.8 3.8 6.5 0.8d 6.9 25.8 5.4 12.5 3.0d
1 10.1 2.9 11.4 16.6 12.8 11.6 23.6 5.9 10.9 14.0 4.4 5.8 0.7d 7.4 25.8 6.2 13.0 3.0d
~~~~
Determined from the fluorescence at 350 nm after tryptic digestion in comparison with the tryptic digest of native BGH. Oxidation was performed at 25" in 0.05 M Tris (pH 8.5) for 5 hr. Oxidation was performed at 25" in perchloric acid (pH 2.85) for 5 hr. Determined from a separate experiment. The proteins were alkylated with iodoacetic acid and then performic acid oxidation was employed to convert the methionine sulfoxide residues to the corresponding sulfone. The methionine values represent the summation of methionine homoserine homoserine lactone. a
Results Oxidation Reaction. The rate of oxidation of methionine residues in BGH under various conditions is illustrated in Figure 1. The rapid oxidation of all four methionine residues was achieved at pH 2.85, while in solutions of pH 8.5 and 11.5 only three out of four residues were affected. In basic media, complete oxidation was achieved only in the presence of 8 M urea. Exclusion chromatography of the product formed in the Tris buffer (without denaturant) on Sephadex G-100 gave a dimeric fraction with a V,/VOratio of 1.79 in 60% overall yield. This compares with a V,/Vo ratio of 1.60-1.65 for the dimer fraction of BGH (Bewiey and Li, 1972). The observed retardation in the elution pattern may be due either to absorption of the protein onto the gel or to a slight shift in equilibrium toward the monomeric form.. All experiments described below were carried out on this dimeric fraction. Amino Acid Analysis. The amino acid analyses (for experimental conditions for hydrolysis, see Glaser et al., 1973) of the oxidized derivatives prepared at pH 2.85 and at 8.5 are given in Table I ; the values are in good agreement with native material. The methionine and methonine sulfoxide contents were determined as follows : the HzOz-treated protein was alkylated with iodoacetic acid and then performic acid oxidation was employed to convert methionine sulfoxide residues to the corresponding sulfone. The content of methionine sulfone as determined by amino acid analysis gave the number of methionine residues which were oxidized by H2O2.The results from this procedure confirmed those from liquid scintillation counting. Three methionine sulfone and 0.7-0.8 methionine residue (methionine plus homoserine plus homoserine lactone) were obtained for each oxidized protein derivative. Identification of the Unreacted Methionine Residue. A single peptide is clearly visible on the autoradiograph. On elution
* The autoradiograph was submitted to the scrutiny of the reviewers and the authors would be glad to forward it to any interested reader.
+
+
and amino acid analysis, the composition3 was found to be Lysl.oVall.oMeto.7. From the reported sequence4 of BGH (Fernandez et al., 1972) and SGH (Li et al., 1972) it is clear the isolated peptide is Val-Met-Lys. Methionine-1 79 is thus resistant to the oxidation in the Tris buffer. Since it has been established that BGH at pH 8.5 exists as a dimer (Bewley and Li, 1972), it was interesting to compare this result to the oxidation at pH 11.5, where the protein is completely in a monomeric form. Autoradiography was carried out exactly as described for the Tris product, and the results were identical. Therefore, in basic media, Met-179 was not available for attack by hydrogen peroxide. On the other hand, in 8 M urea at pH 8.5 or in a solution of pH 2.85, all methionine residues were oxidized (see Figure 1). Rate of Tryptic Digestion. The relative rates of proteolysis of BGH, as measured by alkali uptake, H202-treated BGH (prepared in the Tris buffer), and performic acid oxidized BGH were compared. It was found that the H202-treated 3 The value for methionine is a summation of the values for methionine, homoserine, and homoserine lactone, 'The amino acid sequence in the region of methionine-177 in BGH (Fernandez e? al., 1972) is completely homologous to that of methionine179 in S G H (Li et al., 1972). The discrepancy in numbering is a result of small differences in the reported amino acid sequences and total number of amino acid residues for these proteins. Since the reported sequence of BGH was not complete, positions of amino acid residues are taken from the S G H structure.
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Growth-Promoting Activity of BGH and Its Oxidized Products as Measured by the Rat Tibia Test.
TABLE 11:
2800
z 2400 P 0 .
2000
Preparation
0
1600
BGH BGH-ureaa BGH-Ob
400-i
~,
bo-.
-:
7-T-T-T-TTT---T
2'
4
6
8
10
,
- T - ~,--,-,-r7--,. ~ - ~0i 12 14 16 I8 20 22
'
f
'
~i
24
TIME HOURS
FIGURE 1: Rate of 0.3 M HzOz at 25":
oxidation of methionine residues in BGH by (0)0.05 M Tris (pH 8.5); (0) 0.1 M phosphate (pH 11.5); ( 0 )perchloric acid (pH 2.85); (A) 0.05 M Tris containing 8 M urea (pH 8.5). The kinetics are followed by the subsequent reaction of nonoxidized methionine residues with iodoacetic-l-14C acid in 8 M urea (pH 3.5). protein was digested at about the same rate as native BGH, while performic acid oxidized material was digested at a considerably more rapid rate. Circular Dichroism. The C D spectra of the oxidized BGH at pH 8.2 and the native hormone spectra were identical within experimental error. From the ellipticity values at 221 nm, a-helix contents have been calculated to be approximately 50z. In the region of side-chain absorption, the two spectra were again very similar, showing nearly identical bands. Fluorescence. The relative fluorescence intensity of BGH and its methionine-oxidized derivative in the Tris solvent showed no change in the region of tryptophan fluorescence within experimental error. In order to examine the comparative behavior of these proteins in the presence of a reductant, dithiothreitol was added in large excess (1000-fold over disulfide bridges) and the increase in fluorescence intensity at 330 nm with time was noted (Figure 2). The native material increased fluorescence considerably more slowly than the modified material. The wavelength of maximum fluorescence does not change upon reduction.
5-
o-"
l 4 OI
120
* 10
20
30
40
50
60
70
TIME, MINUTES FIGURE 2: Rate of increase in the fluorescence intensity at 330 nm after addition of 2 mg of dithiothreitol to 2.5 ml of a protein solution (0.14 mg/ml) in 0.05 M Tris (pH 8.5) at 25": (0)BGH; ( 0 ) BGH after oxidation with 0.3 M H202in the same Tris solvent. An immediate quenching effect ( 5 7 3 is observed on addition of the reducing agent.
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BGH-O' Saline
Total Dose (Pg) 20 60 60 20 60 60 0
Tibia Widthd ( p ) 214 i 10 ( 5 ) 270 i. 17 (5) 261 & 23 (4) 227 i 17 (3) 284 i. 11 (5) 211 i 11 (4) 167 & 12(5)
a Treated with 8 M urea as in footnote c in the absence of H2O2. The oxidation was carried out in 0.05 M Tris buffer (pH 8.5) at 25" for 5 hr. The oxidation was carried out as in footnote 6 in the presence of 8 M urea. Expressed as the mean i standarderror of the mean followed by the number of test animals in parentheses.
Biological Studies. Table I1 shows the tibia assay responses. Oxidized hormone prepared in the Tris buffer shows complete retention of its growth promoting activity when compared to native material. Good biological potency was also found when the hormone was incubated with urea; however, oxidation in 8 M urea solution caused a loss of biological potency.
Discussion In Tris buffer of pH 8.5, three out of four methionine residues in BGH were selectively oxidized by HzOp to the corresponding sulfoxide residues. Autoradiographic procedures show that the nonreacting methionine residue was located in position 179 along the peptide chain. Apparently, the region around this methionine was sufficiently hydrophobic to shield this residue from the oxidizing media. Half-cystine residue in position 181 may be an important factor in the lack of accessibility of the neighboring methionine to the solvent. The existence of BGH as a dimer in this buffer does not serve to explain the nonreactivity of methionine-179, as the same results were obtained in the phosphate buffer of pH 11.5 where BGH had previously been shown to exist in a monomeric form (Bewley and Li, 1972). However, solutions of pH 2.85 and 8 M urea did bring about a normalization of methionine residues to the extent that all four methionines were accessible to the oxidizing reagent. Previous studies have demonstrated the many physical, chemical, and biological similarities of BGH and SGH (Bewley and Li, 1972; Glaser et al., 1973; Li et al., 1972; Fernandez et al., 1972). In further support of this view, treatment of SGH with hydrogen peroxide in the Tris buffer (pH 8.5) gave identical results to those obtained with BGH, i.e. three out of four methionine groups were oxidized, and the unreacted methionine residue was shown by autoradiography to be residue 179. The properties of the HzOz-treated BGH (Tris buffer, pH 8.5) were remarkably similar to those of the native hormone. For example, the initial rate of proteolytic digestion has been proposed as a measure of the protein flexibility (Markus, 1965). Protein oxidation, by either hydrogen peroxide or performic acid, does not affect lysine or arginine residues. Therefore, the relative rates of tryptic digestion of BGH and its oxidized derivatives should be a measure of the availability
OXIDATION OF BOVINE GROWTH HORMONE
of peptide bonds for digestion. The equal rate of digestion of BGH and its mildly oxidized derivative indicates that no major conformational change to a less compact form has occurred. This is in contrast to the performic acid oxidized material, which digests at a considerably more rapid rate. The chemical modification of methionine groups does not directly affect chromophoric residues. Therefore, any secondary effects of the modification on the environment of these residues can be probed by a variety of comparative studies, including circular dichroism, ultraviolet spectroscopy, and fluorescence spectroscopy. The amide region of the C D spectra of BGH and its methionine oxidized derivative indicate that the secondary structures of both proteins were essentially identical with an a-helical content of -50 %. Similarly, the close correspondence of bands in the region of side-chain absorption showed the asymmetric microenvironment about the various side-chain chromophores remains unchanged upon oxidation. The absence of any difference spectrum in Tris buffer between BGH and its oxidized derivative was another good indication that the modification had not caused any substantial change in the environment of aromatic residues. Likewise, the identical fluorescence spectra of oxidized BGH in comparison to the native protein were evidence that the chemical modification had neither oxidized tryptophan residues nor resulted in a changed microenvironment around this residue. However, a difference was noted in the fluorescence at 330 nm (Figure 2) when these two proteins were treated with a large excess of dithiothreitol. The greater rate of increase in the derivative is indicative of a loss of rigidity about tryptophan. This may reflect a higher rate of unfolding and/or a higher rate of disulfide bond reduction of the oxidized derivative in comparison to the native hormone. The oxidized hormone in which three out of four methionine residues were modified was fully active as measured by the rat tibia assay. When the fourth methionine residue in position 179 was oxidized in the presence of 8 M urea, the growth promoting potency was diminished (Table 11). This suggests a possible role for Met-179 in the biological activity. In a related investigation on the nature of methionine residues in BGH, Wallis (1972) has presented data showing all four methionine residues are alkylated with excess iodacetic acid in acidic media (0.2 M sodium formate (pH 3.6) with or without 8 M urea). By using iodoacetic-2-14C acid in a twofold molar excess over protein (0.5 pmol of alkylating agent/pmol of methionine), it was also demonstrated that methionine I1 (i.e., methionine residue 124) was considerably less reactive than any of the others, both in the presence and absence of denaturant. These results, in conjunction with our own, show
that in going from acidic to basic media, conformational changes must occur which shield methionine-179 from the reaction media. The experiments described in this report suggest a method currently under investigation for the preparation of protein fragments of BGH and SGH consisting of residues 1-179 and 180-191. Modification of methionine residues with hydrogen peroxide in the Tris buffer can be followed by a cyanogen bromide cleavage at methionine-179 (Gross, 1967). The free methionine residues in the 1-179 fragment can then be regenerated by reduction with thiol reagents. The purified fragments may then be studied by many of the methods described in this article. Acknowledgment The authors thank D. Gordon and J. D. Nelson for technical assistance. References Bewley, T. A., and Li, C . H. (1972), Biochemistry 11,927. Dedman, M. L., Farmer, T. H., and Morris, C. J. 0.R. (1957), Biochem. J . 66,166. Dedman, M. L., Farmer, T. H., and Morris, C. J. 0. R. (1961), Biochem. J . 78,348. Dixon, H. B. F., and Stack-Dunne, M. P. (1955), Biochem. J . 61,483. Fernandez, H. N., Pena, C., Poskus, E., Biscoglio, M. J., Paladini, A. C., Dellacha, J. M., and Santome, J. A. (1972), FEBS (Fed. Eur. Biochem. SOC.)Lett. 25,265. Glaser, C . B., Bewley, T. A., and Li, C. H. (1973), Biochemistry 12,3379. Greenspan, F. S., Li, C. H., Simpson, M. E., and Evans, H. M. (1949), Endocrinology 45,455. Gross, E. (1967), Methods Enzymol. 11,238. Li, C. H. (1954), J. Biol. Chem. 204, 391. Li, C. H., Dixon, J. S., Gordon, D., and Knorr, J. (1972), Znt. J . Protein Res. 4,151. Markus, G. (1965), Proc. Nut. Acad. Sci. U . S . 54,253. Neher, R., Riniker, B., Maier, R., Byfield, P. G. H., Gudmundsson, T. V., and MacIntyre, I. (1968), Nature (London) 220,984. Papkoff, H., and Li, C. H. (1958), Biochim. Biophys. Acta 29, 145. Tashjian, A. H., Jr., Ontjes, D. A,, and Munson, P. L. (1964), Biochemistry 3, 1175. Wallis, M. (1972), FEBS (Fed. Eur. Biochem. SOC.) Lett. 21,118.
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